The developments of genomic techniques provide a great deal of basic information at gene level for the investigation of biological functions, diagnosis and treatment of disease, and development of medicaments etc. But for most of the biological systems, the gene information alone is not sufficient for illustrating the function mechanism thereof. Furthermore, if these information as well as the function mechanism are to be used for the purposes of applications, such as identification and analysis for pathological conditions, and verification and determination for the function mechanism of medicaments, then it will be necessary to further obtain more information at the protein level, such as analysis and investigation for various protein propertied like the structure, the function, the expression, the localization, the modification, and the interaction etc.
The identification for various properties of a protein will need many technical means, such as MS, chromatography, electrophoresis, chip, and various labeling techniques. Many of these technical means will need to be carried out based on antigen-antibody interactions. Further, antigen-antibody interaction per se is also an important technical means, which can be widely used in various fields like scientific researches, medical treatment, and medicament development, such as the development of therapeutic antibody medicament etc. With the development of protein researches, the demand for various antibodies as well as antibody libraries is increasing. For example, it could be necessary to prepare a specific antibody library against all the proteins in the proteome of a specie, or to prepare specific antibodies against a particular type of proteins like kinases or G-protein receptors. However, only limited amount of antibodies against several thousands of proteins are known in the art, and the specificities thereof are not sufficient for the requirements of many technical means. Therefore, it would be important to rapidly and effectively prepare large amount of antibodies against any protein of interest.
Currently, the commonly used methods for obtaining antibodies include hybridoma techniques, recombinant antibody techniques, various molecular display techniques, and the combination of these techniques with high-throughput techniques.
For the preparation of antibodies, generally a native or recombinant protein or fragment thereof is used to immunize an animal, so that an antibody that can specifically recognize and bind the protein is produced in the animal. Then various technical means can be used based on corresponding requirements to obtain antibody from the animal, such as monoclonal antibody or polyclonal antibody. The production of monoclonal antibody will typically rely on hybridoma techniques. In such techniques, after immunizing the animal, the cells of the animal will be taken and fused to generate an antibody-producing hybridoma, which will then be cloned to construct a strain for producing antibody, and subsequently the antibody will be purified and identified. The antigen's epitope for the antibody can also be further determined according to the requirements. Such hybridoma techniques for producing monoclonal antibodies were first applied in mouse model (Köhler and Milstein, Nature vol. 256, 1975). Currently they are widely used in various animal models, and the detailed procedure thereof can be seen in various textbooks and operation manuals (such as, Bazin, “Rat hybridomas and rat monoclonal antibodies”, CRC Press, 1990; Goding, “Monoclonal antibodies: principles and practice”, 3rd edition, Academic Press, 1996; Shepherd and Dean “Monoclonal antibodies” Oxford University Press, 2000 etc.). Although these methods currently are widely used in the preparations of antibodies, they also have many disadvantages, such as very long preparation periods, very complicated preparation techniques, incomplete recognition of epitopes, and high cost etc. Further, such methods cannot be used for all the proteins, e.g. for many antigens with low immunogenicity or antigens with toxicity, such methods would be inappropriate (Sinclair N R (et al, 2004) B cell/antibody tolerance to our own antigens. Front Biosci 9: 3019-3028).
Furthermore, in order to obtain monoclonal antibodies with specificity, generally, a chemical synthesized peptide fragment is coupled to a carrier protein, which is then used to immunize a mouse. Such a method can generate an antibody against a single epitope of one protein. But due to the differences in the immunogenicity of different fragments, the overall success rate of such a strategy is relatively low, and especially for proteins with high homology, the fragments of which have poor immunogenicity and can hardly stimulate the mouse to produce potent immune responses. Another commonly used strategy is to produce the immunogen with full length protein or protein fragment, which can partially solve the above problem; but there still exists an disadvantage of poor overall success rate for protein expression (30-70% for commonly used expression and purification systems)(Thorsten Kohl, Christian Schmidt, Stefan Wiemann, Annemarie Poustka and Ulrike Korf. Drew, 2003). For protein fragments with high homology with the proteins of the animal used as model, the immune responses in the animal are generally very weak, and thus the success rate for preparing monoclonal antibody is quite low (Sinclair N R et al, 2004, Automated production of recombinant human proteins as resource for proteome research Proteome Science 2008, 6:4; Sinclair N R (2004) B cell/antibody tolerance to our own antigens. Front Biosci 9: 3019-3028).
The techniques of recombinant antibody can be various molecular display techniques, so as to produce antibodies (with high affinity to the target) against several antigens, and the antigen epitopes can also be simultaneously determined. Therefore, they are commonly used in the development of medicaments (Christine Rothe, Stefanie Urlinger, Makiko Yamashita et al. The Human Combinatorial Antibody Library HuCAL GOLD Combines Diversification of All Six CDRs According to the Natural Immune System with a Novel Display Method for Efficient Selection of High-Affinity Antibodies. J. Mol. Biol. (2008) 376, 1182-1200, 2007). However, the operation of the techniques of recombinant antibody is complicated, the cost thereof is high, and the yield thereof is relatively low. Further, there often exists non-specific binding. Therefore the large scale application of such techniques is limited. (Levitan, B. Stochastic modeling and optimization of phage display. J. Mol. Biol. 277, 893-916 (1998). Bradbury et al, 2004)
In order to increase the efficiency of immunization and screening, the above techniques can be combined with high-throughput methods, such as the high-throughput strategy in which several immunogens are simultaneously used for the immunization and chip techniques are used for the screening, e.g. as described in CN200510026873.0. However, such immunization strategy will require a great amount of immunogens with high immunogenicity. This can hardly be accomplished for proteins that are difficult to be expressed, or for proteins with very low immunogenicity.
Furthermore, in conventional immunization methods using several immunogens, the epitope that the produced antibody is directed to can only be passively determined based on the requirements using particular techniques such as epitope mapping after the antibody is produced (see, Glenn E. Morris, “Methods in Molecular Biology: Epitope Mapping Protocols”, Humana Press, 1996). Sometimes the epitope is not unique for the protein of interest, and can present in many other proteins, such that the specificity of the produced antibody is relatively low.
In order to solve the various problems mentioned above, new methods for preparing and screening antibodies are desired, so as to effectively and rapidly prepare and screen high specific antibodies against all the proteins with low cost.